Rupture of atherosclerotic plaques in the carotid artery results in neurological problems. Inflammatory cytokines in the plaque induce apoptosis of vascular smooth muscle cells (VSMCs), thus thinning the fibrous cap and resulting in plaque destabilization and rupture. During the initial funding cycle, we made novel observations that IGF-1 is more potent in inducing the survival of VSMCs in carotid endarterectomy tissues of asymptomatic than symptomatic patients. Also, atheroma-associated cytokines decrease IGF-1-induced survival and proliferation of VSMCs. In our follow-up studies we observed specific differences in the profile of dendritic cells (DCs), T-regulatory cells, and NPY and NPY receptors in symptomatic (unstable) and asymptomatic (stable) plaques. Also, stronger immunoreactivity of Foxp3, IL-10 and CD25 with preponderance of CD4? T cells in carotid plaques of asymptomatic than symptomatic patients. IGF-1 increased mRNA transcripts of NPY-Y2 and NPY-Y5 receptors in asymptomatic but not in symptomatic plaque SMCs. These novel findings suggest a cell-mediated immune response in carotid plaques which is regulated by NPY. Our central hypothesis is that the inflammation in the carotid plaque is modulated by interaction of NPY with DCs and T cells leading to increased inflammation and apoptosis of VSMCs in carotid plaques of symptomatic patients with carotid stenosis, and that atheroma-associated cytokines modulate NPY receptors and their function in VSMCs of carotid plaques. In Aim 1, we will examine the phenotypes of dendritic cells and the effect of NPY, in the presence and absence of IGF-1, on the cell surface expression of co-stimulatory molecules in DCs in the carotid plaques of symptomatic and asymptomatic patients with carotid stenosis. In Aim 2, we will examine the phenotype of T-regulatory cells (inducible CD4? T-regs and naturally-occurring CD4???? T-regs) and the effect of NPY, in the presence and absence of IGF-1, on the cell surface expression of the receptors for co-stimulatory molecules (PD-1, CTLA- 4) in T-regs from the carotid plaques of symptomatic and asymptomatic patients with carotid stenosis. We will also determine the effect of NPY, in the presence and absence of atheroma-associated cytokines, on the differentiation of naive T cells into T-regulatory cells. In Aim 3, we will examine the effect of NPY, in the presence and absence of inflammatory cytokines, on the survival and apoptosis of VSMCs isolated from the carotid plaques of symptomatic and asymptomatic patients with carotid stenosis. The long-term goal of this study is to determine the cellular and molecular mechanisms responsible for the instability and rupture of carotid plaques in patients with carotid stenosis. Such investigations would provide unique insights to the pathophysiologic process of neurological diseases associated with the rupture of atherosclerotic plaques and the means to prevent the disease in patients with carotid stenosis. PUBLIC HEALTH RELEVANCE: Many patients with blockade of their neck artery develop neurological symptoms including transient ischemic attack or stroke. This is primarily due to breaking off the atherosclerotic plaque in the neck artery. In this project experiments are proposed to examine the underlying cellular and molecular mechanisms of the plaque rupture. Information obtained from this study should provide an opportunity to formulate superior therapeutic approaches in the prevention of stroke.